PROJECT SUMMARY ! Asbestosis is a prototypical fibrotic lung disease with several pathological features common to fibrosis in other organ systems. Asbestos exposure remains to be a hazard to workers and results in 100,000 deaths in the U.S. annually. There are no accepted therapies for asbestosis, and recently approved anti-fibrotic therapies have limited efficacy. The generation of mitochondrial ROS (mtROS) from alveolar macrophages plays an integral role in the pathogenesis of pulmonary fibrosis by increasing TGF-?1 production. The source and molecular mechanism(s) that regulate mtROS in alveolar macrophages are not known. One contribution to mtROS generation is calcium (Ca2+) influx into the mitochondria. Ca2+ entry from the endoplasmic reticulum (ER) and cytosol is regulated by the mitochondrial Ca2+ uniporter (MCU). The unifying theme of this application is that mtROS is present in alveolar macrophages during fibrotic repair, and the increase in macrophage- derived TGF-?1 is, in part, regulated by Ca2+ entry into the mitochondria via MCU. Recent important data from our lab show that alveolar macrophages are a critical source of TGF-?1 in the lung, and the alveolar macrophages that produce TGF-?1 are apoptotic resistant. These macrophages are also pro-fibrotic, which may contribute to their prolonged survival. Importantly, our data demonstrates that MCU expression in vivo increases active TGF-?1 in BAL fluid, whereas MCU-deficient mice have macrophages that are anti-fibrotic and undergo apoptosis. The role of MCU and mitochondrial Ca2+ in any cell type has not been linked to pulmonary fibrosis. The hypothesis of this application is that MCU induces an apoptosis-resistant, pro-fibrotic alveolar macrophage that contributes to asbestos-induced pulmonary fibrosis. We will test this hypothesis with three specific aims. Aim 1 will determine if macrophage MCU and mitochondrial Ca2+ are linked to fibrosis development using conditional macrophage DN-MCU transgenic mice. Aim 2 will determine if modulation of MCU and mitochondrial Ca2+ regulates alveolar macrophage apoptosis resistance. Aim 3 will determine if modulation of MCU in alveolar macrophages of asbestosis patients alters the pro-fibrotic phenotype and apoptosis resistance. The studies are innovative because understanding the role of MCU in alveolar macrophages may provide an important target for preventing or halting progression of fibrosis development.